15-oxygenated-delta3-a-norandrostene-2, 17-diones



United States Patent Ofi 3,367,963 Patented Feb. 6, 1968 ice Thisinvention relates to and has as its object the provision of novelphysiologically active steroids, and methods for their production.

More particularly, this invention relates to the production of compoundsof the formula wherein R is hydrogen; R is selected from the groupconsisting of hydroxy and acyloxy; and together R and R is oxo i Thepreferred acyloxy radicals are those of hydrocarbon carboxylic acids ofless than twelve carbon atoms, as exemplified by the lower alkanoicacids (e.g., acetic, propionic, butyric and tert-pentanoic acid), thelower alkenoic acids, the monocyclic aryl carboxylic acids (e.g.,benzoic and toluic acid), the monocyclic aryl lower alkanoic acids(e.g., phenacetic and fl-phenylpropionic acid), the cycloalkanecarboxylic acids and the cycloalkene carboxylic acids.

The compounds of the instant invention are physiologi cally activesteroids which possess anti-androgenic activity, i.e., they inhibit theaction of androgens, and they may be used in the treatment of suchconditions as hyperandrogenic acne. The compounds may be formulated forsuch administration, the concentration and/or dosage being based on theactivity of the particular compound and the requirements of the patient.

The novel compounds of this invention may be prepared according to theprocess of this invention by employing as the starting material, A-A-norandrostene-2,l7- dione.

The novel compounds of the instant invention may be prepared from thestarting material by subjecting the latter to the action of the enzymesof Collectotrichum linz'cola under oxidizing and preferably aerobicconditions. The oxidation can best be effected by either including thestarting material in an aerobic culture of the microorganism, or bybringing together, in an aqueous medium, the compounds, air, and enzymesof non-proliferating cells of the microorganism.

In general, the conditions of culturing the microorganism for thepurpose of this invention are (except for the inclusion of the startingmaterial to be converted) the same as those of culturing microorganismsfor the production of antibiotics, i.e., the microorganism isaerobically grown in contact with (in or on) a suitable fermentationmedium. A suitable medium essentially comprises a source of carbon andenergy. The latter may be a carbohydrate (such as sucrose, molasses,glucose, maltose, starch, or dextrin), a fatty acid, a fat and/or thecompound itself. Preferably, however the medium includes an assimilablesource of carbon and energy in addition to the steroid. Among the fatsutilizable for the purpose of this invention are: lard oil, soybean oil,linseed oil, cottonseed oil,

peanut oil, coconut oil, corn oil, castor oil, sesame oil, crude palmoil, fancy mutton tallow, sperm oil, olive oil, tristearin, tripalmitin,triolein, and trilaurin. Among the fatty acids utilizable for thepurpose of this invention are: stearic acid, palrnitic acid, oleic acid,linoleic acid, and myristic acid.

The source of nitrogenous factors may be organic (e.g., soybean meal,corn steep liquor, meat extract and/ or distillers solubles) orsynthetic (i.e., composed of simple, synthesizable organic or inorganiccompounds such as ammonium salts, alkali nitrates, amino acids or urea).

An adequate sterileair supply should be maintained during fermentation,for example, by the conventional methods of exposing a large surface ofthe medium to air or by utilizing submerged aerated culture. Thecompound may be added to the culture during the incubation period, orincluded in the medium prior to sterilization or inoculation. Thepreferred (but not limiting) range of concentration of the startingmaterial is about 0.01 to 0.10%. The culture period (or rather the timeof su-b-- jecting the compound to the action of the enzyme) may varyconsiderably, the range of about six to ninety-six hours being feasible,but not limiting.

The process yields, inter alia, A -A-norandrostene-15aol-2,17-dione. TheA -A-norandrostene-15a-ol-2,17-dione can be esterified in the usualmanner, as by treatment with the desired acid anhydride or acy] halidein an organic solvent (preferably in an organic base such as pyridine)to yield the ISa-ester derivative, or it may be oxidized to yield theIS-keto derivative.

The following examples are illustrative of the invention:

Example I .]5a-hydr0xy-A -A-n0randr0stene-2,17-di0ne (A)Fermentation-Surface growth from each of two 2 week old agar slants ofco lletotrichum linicola (NCTC- 1194), (National Collection TypeCulture, London, England), the slants containing as a nutrient medium(A):

Grams Glucose 1O Yeast extract 2.5 K HPO, 1 Agar 20 Distilled water toone liter.

is suspended in 5 ml. of 0.01% aqueous sodium lauryl sulfate solution.One ml. portions of this sunpension are used to inoculate eight 250 ml.Erlenmeyer flasks, each containing 50 ml. of the following sterilizedmedium (B):

Distilled water to one liter.

After 96 hours incubation at 25 with continuous rotary agitation (280cycles/minute; two inch radius), 10% (-vol./vol.) transfers are made tothirty-four 250 ml. Erlenmeyer flasks each containing 50 ml. of freshlysterilized medium B. After 24 hours of further incubation, using thesame conditions as described above, the steriod (300 micrograms/ml.) isadded by supplementing each flask with 0.25 ml. of a sterile solution(60 ing/ml.) of A A norandrostene-2,17-dione in N,N-dimethylformamide. Atotal of 510 mg. is fermented.

After approximately nine hours of further incubation, using identicalconditions as described above, the contents of the flasks are pooled andthe broth is then filtered through a Seitz clarifying pad. The flasks,mycelium and pad are washed with successive 50 ml. portions of warm TMS533 233 m (16,100); enor.

9.04 s, l8-Me) 8.79 (s, l9-Me), 5.53 (m, ISB-H) and 4.27 (s, 3-H).

Analysis.Calcd. for C H O (288.37): C, 74.97; H,

8.39. Found: C, 74.85; H, 8.44.

Example 2.-15a-acet0xy-A -A-nrandr0stene-2,1 7 -di0ne A mixture of 35mg. of a-hydroxy-A -A-norandrostene-2,17-dione, 0.25 ml. of aceticanhydride and 0.5 ml. of pyridine is left at room temperature for 4.25hrs, diluted with water and extracted three times with ether. The etherextracts are washed with a saturated sodium bicarbonate solution, 8%salt solution, dried over sodium sulfate and evaporated to dryness. Theresidue is crystallized from ether to give about mg. of 15a-acetoxy-AA-norandrostene-2,17-dione having a melting point of about 153-455". Theanalytical sample is prepared by recrystallization from ethylacetate-isopropyl ether, M.P. about 153-155", [a] -+63 (EtOH); A 5.75,5.95 and 6.1812;

TMS

85,12? 232 111;]. (18,300); T013013 9.00 (s, 18-Me), 8.80 (s, 19-Me),4.78 (111, 156-11) and 4.26 (s, 3-H).

Analysis.-Calcd. for C l-1 0 (330.41): C, 72.70; H, 7.93. Found: C,72.80; H, 791.

Similarly, by substituting other acylating agents, such as propionicanhydride and benzoyl chloride, for the acetic anhydride, thecorresponding esters are formed.

Example 3.-A -A-n0randr0stene-2,15,17-tri0ne A solution of mg. oflSa-hydroxy-M-A-norandrostene-2,17-dione in 9 m1. of acetone is treateddropwise with an equivalent amount of chromium trioxide-sulfuric acid.The chromic sulfate is removed by filtration, and washed with additionalacetone. The filtrate is concentrated, diluted with water and extractedWith chloroform. The chloroform extracts are washed with 8% saltsolution, dried over sodium sulfate and evaporated to dryness to give A-A-norandrostene-2,15,17-trione.

The invention may be variously otherwise embodied within the scope ofthe appended claims.

What is claimed is:

1. A compound of the formula References Cited UNITED STATES PATENTS10/1965 Weisenborn 260-488 12/1966 Fried et al 260-488 LORRAINEA.WEINBERGER, Primary Examiner.

V. GARNER, Assistant Examiner.

1. A COMPOUND OF THE FORMULA